The present invention relates to the chemical synthesis of biopolymers, and specifically, to a device for the simultaneous synthesis of large numbers of biopolymers, for example, polynucleotides, polypeptides and polysaccharides. The development of methods for the chemical synthesis of biopolymers of any desired sequence has resulted in great advances in many areas of biology and medicine during recent years. For example, physical and biochemical studies of the structure and interactions of synthetic DNA fragments has led to important new findings concerning the molecular mechanisms of genetic processes, including DNA metabolism, and regulation of gene expression. Synthetic polynucleotides have played a key role in studies of genetic organization through their use as primers for DNA sequencing and as hybridization probes, linkers and adapters in the cloning of genes. of genes. An additional use of synthetic polynucleotides is in DNA probe technology in the diagnosis of disease. Ultimately, synthetic polynucleotides may be used in gene replacement therapy to cure genetic disorders, and in other genome engineering procedures to provide resistance to disease and starvation. Synthetic polynucleotides are routinely used for site-directed in vitro mutagenesis, for studying the structure-function relationships within genetic regulatory elements and for determining the effects of specific amino acid substitutions on the functions of proteins. The latter use, termed protein engineering, will not only facilitate an understanding of the mechanism of action of enzymes and other proteins, but will also permit the design of functionally superior proteins and drugs, leading to important advancements in medicine, agriculture and industry. Likewise, the availability of synthetic defined-sequence polypeptides is bringing about equally dramatic advancements in protein chemistry, immunology, pharmacology and biotechnology.
In many genetic engineering projects it is necessary to use a large number of differently defined sequence polynucleotides, sometimes hundreds of different sequences in a single experiment. Similarly, some protein chemistry experiments require hundreds of different peptide sequences. In order to determine the nucleotide sequence of the human genome millions of different polynucleotide primers will be required. The latter endeavor, along with many other worthwhile projects that could be carried out by individual laboratories, are economically impractical with the current cost to the investigator of synthetic polynucleotides ($5-$10 per nucleotide residue).
The capability to chemically synthesize polynucleotides of defined sequence resulted from the pioneering work of Michelson and Todd in the 1950s, (Michelson, A.M. & Todd, Sir 20 A. R., "Nucleotides Part XXXII. Synthesis of a Dithymidine Dinucleotide Containing a 3':5' Internucleotide Linkage," J. Chem. Soc.-1955, pp. 2632 2638), in which a method was developed for specific chemical synthesis of 5'-3' internucleotide phosphodiester linkages. This procedure was developed further over the next 20 years, culminating in the total synthesis of a gene for transfer RNA by Khorana and Associates, (Khorana, H.G., "Total Synthesis of a Gene," Science, Vol. 203, pp. 614-625, (1979). Recently, the phosphate diester method has been replaced by the phosphate triester method (Letsinger, R.L. and Ogilvie, K.K., "A Convenient Method for Stepwise Synthesis of Oligothymidylate Derivatives in Large-Scale Quantities," J. Am. Chem. Soc., Vol. 89, pp. 4801-4803, (1967); Narong, S.A., Brousseau, R., Hsiung, H.M. and Michniewicz, J.J. "Chemical Synthesis of Deoxyoligonucleotides by the Modified Triester Method, Meth. Enzymol, Vol. 65, pp. 610-620, (1980)) and the phosphite triester method (Letsinger, R.L., Finnan, J.L., Heavener, G.A. and Lunsford, W.B., "Phosphite Coupling Procedure for Generating Internucleotide Links," J. Am. Chem. Soc., Vol. 97, pp. 3278-3279, (1975); Beaucage, S.L. and Caruthers, M.H., "Deoxynucleotide Phosphoramidites--A New Class of Key Intermediates For Deoxypolynucleotide Synthesis," Tet. Lett., Vol. 22, pp. 1859-1862, (1981)), which have the advantage of more rapid synthesis and fewer side reactions. Both of these methods can be carried out in solution as originally devised, and have been adapted for solid phase synthesis of polynucleotides (Matteucci, M.D. and Caruthers, M.H., "Synthesis of Deoxyoligonucleotides on a Polymer Support," J. Am. Chem. Soc., Vol. 103, pp. 3185-3191, (1981); Sproat, B.S. and Bannwarth, W., "Improved Synthesis of Oligodeozynucleotides On Controlled Pore Glass Using Phosphotriester Chemistry and a Flow System," Tet. Lett., Vol. 24, pp. 5771-5774, (1983)). Solid phase synthesis offers the advantage of greater speed of synthesis because the growing chain is covalently attached to an insoluble support, permitting reagents to be washed away between chemical steps and obviating the need to purify the polynucleotide product after each addition of monomer. Furthermore, solid phase synthesis permits automation of the process, so that each base addition (via multistep reaction cycle) can be carried out in about ten minutes at ambient temperature. The high efficiency of condensation under these conditions (currently 99%) permits the automated synthesis of polydeoxynucleotides of chain length greater than 100.
Chemical procedures used for solid phase synthesis of polypeptides are frequently based on the original protocol of Merrifield, which was successfully used for synthesis of enzymically active, 124-residue ribonuclease A (Gutte, B. and Merrifield, R.B., "The Synthesis of Ribonuclease A," J. Biol. Chem., Vol. 246, pp. 1922-1941, (1971)). This procedure uses standard polystyrene-divinylbenzene supports, t-butyloxycarbonyl (Boc) amino group protection, and DCC-activated condensation with symmetric anhydride intermediates. The procedure has been used successfully in automated peptide synthesizers, as well as in the multiple simultaneous synthesis method of Houghton described below.
Several alternate procedures for peptide synthesis have been devised. One particularly advantageous one (Auffret, A.D. and Meade, L.G., "Alternative Strategies in Peptide Synthesis," Synthetic Peptides in Biology and Medicine, Alitalo, K., Partanen, P. and Vaheri, A. (Eds.), Elsevier Science Publishers, Amsterdam, 1985) utilizes a composite polyamide-Kieselguhr support (found to be superior for continuous flow synthesis), fluorenylemethozycarbonyl (Fmoc) amino group protection, and 1-hydrozybenzatriazole-activated condensation with pentafluorophenyl ester (PFPE) intermediates. The high stability of the active ester intermediates make them more conveniently used for peptide synthesis than the relatively unstable anhyride intermediates.
Recent developments in polynucleotide synthesis, including descriptions of the chemical reactions, are summarized in review articles by John Smith ("Automated Solid Phase Oligodeoxyribonucleotide Synthesis", American Biotechnology Laboratory, pp. 15-24 (December 1983)) and Marvin Caruthers ("Gene Synthesis Machines: DNA Chemistry and Its Uses", Science, Vol. 230, pp. 281-85 (1985)). One particularly promising recent advancement is the development of cost effective procedures for in situ generation of phosphoramidite intermediates from inexpensive protected nucleosides (Barone, A.D., Tang, J.Y. and Caruthers, M.H., "In Situ Activation of Bis-Dialkylaminophosphines--A New Method for Synthesizing Deoxyoligonucleotides on Polymer Supports," Nucl. Acids Res., Vol. 12, pp. 4051-4061, (1984); Nielsen, J., Taagaard, M., Marigg, J.E., van Boom, J.H. and Dahl, 0., "Application of 2-cyanoethyl N, N, N', N'-tetraisopropylphosphorodiamidite for In Situ Preparation of Deolyribonucleoside Phosphoramidites and Their Use in Polymer--Supported Synthesis of Oligodeoxyribonucleotides," Nucl. Acids Res., Vol. 14, pp. 7391-7403, (1986)).
Another advantageous recent development is the use of amidine groups to protect exocyclic amino groups (e.g., Caruthers, M.H., McBride, L.J., Bracco, L.P. and Dubendorff, J.W., "Studies on Nucleotide Chemistry 15. Synthesis of Oligodeoxynucleotides Using Amidine Protected Nucleosides," Nucleosides and Nucleotides, Vol. 4, pp. 95-105, (1985)). Amidine protecting groups stabilize deolyadenosine residues against acid-catalyzed depurination, which occurs during the detritylation step of the synthesis cycle, thereby permitting synthesis of longer polynucleotides.
Finally, a procedure for synthesis of RNA polymers on silica supports, involving a modified phosphoramidite approach, has recently been reported (Kierzek, R., Caruthers, M.H., Longfellow, C.E., Swinton, D., Turner, D.H. and Freier, S.M., "Polymer-Supported RNA Synthesis and its Application to Test the 5 Nearest--Neighbor Model for Duplez Stability," Biochemistry, Vol. 25, pp. 7840-7846, (1986)).
Although the above methods permit the synthesis of one or a few polynucleotide sequences at a time, at moderate cost, there is a great need for technological development in this area, to reduce the cost of synthesis and to permit simultaneous synthesis of large numbers of polynucleotide sequences. Progress toward this aim has recently been made in the form of procedures and devices that permit multiple simultaneous synthesis of polynucleotides or polypeptides.
Frank et al. ("A New General Approach for the Simultaneous Chemical Synthesis of Large Numbers of Oligonucleotides: Segmented Solid Supports", Nucleic Acid Research, Vol. 11, No. 13, pp. 4365-77 (1983)) recently used cellulose filters as a solid phase support for polynucleotide synthesis. A protected nucleoside was covalently linked to the hydroxyl groups of the filter paper by 3'-o-succinate linkage, then elongated by the phosphate triester procedure used previously with loose beaded solid phase support materials. Frank et al. reported synthesis of two octamers, and proposed that by stacking the paper filters into four different reaction columns, designated for addition of A, G, C and T residues to the growing chain and sorting the discs between reaction cycles, a large number of different polynucleotide sequences could be simultaneously synthesized. Frank, et al. demonstrated that the two octamers synthesized by this procedure (present within the same reaction column during most cycles) were obtained at reasonable yield, and DNA sequence analysis proved that the products consisted of the expected nucleoside sequences and were not contaminated by each other.
The proposed use of the filter paper method for simultaneous synthesis of many sequences was later implemented by Matthes et al. ("Simultaneous Rapid Chemical Synthesis of Over One Hundred Polynucleotides on a Microscale", The EMBO Journal, Vol. 3, No. 4, pp. 801-05 (1984)). These authors used a phosphate triester procedure similar to that reported by Frank et al., to simultaneously synthesize over one hundred polynucleotide sequences within a period of two weeks. Several limitations of the Matthes et al. procedure exist. Due to low loading capacity of the filter paper disks, their hydrophilic nature and their unfavorable mass transfer properties, the coupling efficiency at each step is poor compared with that attained with the standard solid phase synthesis procedures, and only a very small quantity of desired polynucleotide is produced, of limited chain length (up to about 20-mer). The product is heavily contaminated by shorter failure sequences, and must be purified by time-consuming procedures before use. Nevertheless, this procedure has the potential of yielding large numbers of sequences at low cost. This method apparently has been attempted by many laboratories, but apparently only a very few laboratories have been able to obtain usable products using the technique.
Another report (Bannwarth, W. and Laiza, P., "A System for the Simultaneous Chemical Synthesis of Different DNA Fragments on Solid Support," DNA, Vol. 5, pp. 413-419, (1986)) describes a mechanical apparatus that can simultaneously synthesize several different polynucleotides. The device consists of a series of stackable rotatable metal disks, each containing, in radially symmetrical position, a single reaction chamber plus a number of narrow "bypass" holes. The stacked metal disks can be rotated to produce vertical alignment of all reaction chambers designated for addition of a given nucleoside residue to the support-bound DNA chains contained therein, with these chambers being connected by bypass holes. Thus, by appropriate rotation of the metal disks following each reaction cycle (created by sequential flow of reagents and solvents through the system), a different DNA sequence is synthesized for each of the stacked metal disks. The chief advantage of this device over the segmented filter paper method is higher coupling efficiency, enabled by the placement of controlled pore glass supports within the reaction chambers. DNA chains of up to 36 residues long were produced utilizing phosphoramidite chemistry. Another advantage of the design is its potential for automation. The chief disadvantage is the relatively low number of simultaneous synthesis (a maximum of 12 DNA fragments were simultaneously produced).
In another approach, utilized for simultaneous synthesis of different polypeptides, (Houghten, "General Method for the Rapid Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction of the Level of Individual Amino Acids", Proc. Natl. Acad. Sci. USA, Vol. 82, pp. 5131-35 (August 1985)), Houghten employed polypropylene mesh bags containing solid phase support resins used for standard solid phase synthesis of peptides. By placing a number of these resin-containing bags into a single stirred reaction chamber, all peptide sequences to which a given amino acid was to be added could undergo the coupling reaction simultaneously. The authors used this procedure to simultaneously synthesize 248 different 13-mer peptides which were obtained in yield comparable to that attained by standard single-peptide solid phase methods. In this work, each of the 13-mer peptides actually consisted of a sequence identical to the "control sequence," except for a single amino acid replacement. Thus, at each amino acid addition, the vast majority of the resin-containing bags were placed into the same stirred reaction vessel, while only those resins containing peptides to which a unique amino acid was to be added at that position in the sequence were reacted separately from the bulk of the material. Although the "different" peptide sequences synthesized in Houghten's original work each consisted of the same sequence, with a single amino acid change from the "control sequence," it was proposed that by use of a multiplicity of stirred reaction vessels, each containing many resin-containing bags, the procedure could be used for simultaneous synthesis of a large number of completely unique peptide sequences. Houghten's "tea bag" method, including description of its use for simultaneous synthesis of 120 entirely different 15-residue peptides, is further described in Houghten et al, "Simultaneous Multiple Peptide Synthesis: The Rapid Preparation of Large Numbers of Discrete Peptides for Biological Immunological, and Methodological Studies," Biotechniques, Vol. 4, No. 6, pp. 525-28 (1986).
Two difficulties may prevent the Houghten "tea bag" method from being implemented for simultaneous synthesis of large numbers of polynucleotide sequences. The sealable polypropylene mesh bags are not sufficiently inert to be used in the phosphate triester and phosphite triester procedures currently used for polynucleotide synthesis. Support containing porous bags constructed of inert materials such as TEFLON are difficult, if not impossible to seal, making it difficult to prevent loss of solid phase support from the bags during synthesis. A more serious problem is that in the solid phase procedure for polynucleotide synthesis, sufficient space must be left in the column above the support bed, such that as solvents and reagents are pumped from below, the support is "lifted" by the upward flow, resulting in the necessary mass transfer within the beads required for nearly quantitative chemical reactions. The physical properties of the non-rigid "tea bags" would not permit the necessary lifting of support materials during passage of solvents and reagents through the column.
Accordingly, due to the shortcomings of the present devices and procedures, there exists a need for a device and procedure for rapid, simultaneous synthesis of large numbers of any biopolymer of different sequences at high yields and lower costs.